The present invention relates to ligand/anti-ligand assays for detection and measurement of adherent proteins, including lipophilic serum and plasma proteins, cytokines, globular serum and plasma proteins, and pentraxins. Assays of the present invention are particularly useful for detection and measurement of serum amyloid A, apolipoprotein Al, apolipoprotein B, CRP, IL-1 beta, TNF alpha, albumin and similar adherent proteins.
Biological fluids such as plasma and serum contain numerous proteins. The presence, absence or concentration of a particular protein may be of interest because, e.g., such data provide information regarding the clinical state of the individual from which the biological fluid was obtained. Accordingly, it is desired to have relatively simple and inexpensive assays to detect the presence of and determine the concentration of such proteins.
By way of example, plasma and serum contain several classes of proteins which are non-covalently linked to lipids. These lipophilic proteins perform a variety of functions, including lipid transport, intercellular communication, and host defense. Medical research has found that in a number of disease states, levels of lipophilic serum and plasma proteins deviate from those found in non-disease states. The significance of these deviations is the subject of active clinical research.
The concentrations of many of the lipophilic serum or plasma proteins change as the physical state of the body changes. For example, the level of serum amyloid A dramatically increases during inflammation; the level of apoliprotein Al decreases during coronary disease, while that of apolipoprotein B is elevated during coronary disease. Clinical measurement of these kinds of proteins will therefore become more important as more is learned about the mechanisms of various disease states.
Accurate and reproducible measurement of lipophilic serum or plasma proteins has historically been difficult. Many factors contribute to these difficulties, such as the existence of multiple forms of the lipophilic proteins and the inherent "stickiness" of the lipophilic proteins.
Organisms frequently contain several molecular forms of a particular lipophilic protein, which differ only slightly in amino acid sequence. These molecular forms are known as isotypes, each of which may in turn may exist in multiple conformational states. Immunological reactivity may vary among isotypes and conformational states of a particular isotype.
Conformational changes are frequently observed in apolipoproteins which accompany the binding of lipid by the apolipoprotein. (Bausserman et al, J. Biol. Chem. 258, 10681 (1983); Segrest et al., Biochemistry 15, 3187 (1976); Segrest et al., FEBS Letters 38, 247 (1974); Morrisett et al, Biochemistry 12, 1290 (1973)). It is possible to observe these conformational changes when the protein in serum or plasma is analyzed by immunodiffusion or charge shift immunoelectrophoresis (Linke, Biochim. Biophys. Acta 668, 388 (1981)). Treatment with heating, acid, alkali, guanidine hydrochloride and extraction with organic solvents can also induce changes in apolipoprotein conformation. (Sipe et al., Br. J. Exp. Path. 57, 582 (1976); Pepys and Baltz, Adv. Immunol. 34, 141 (1983); Eriksen and Benditt, Meth. Enzymol. 128, 311 (1986); Maciejko, Clin. Chem. 28, 199 (1982)).
Moreover, lipophilic proteins are "sticky" , i.e., they form non-specific hydrophobic interactions with other molecules of the same structure (also referred to as self association). This stickiness also causes non-specific hydrophobic interactions between the lipophilic proteins and unrelated serum proteins and laboratory vessels. (Franklin, J. Exp. Med. 144, 1679 (1976); Marhaug and Husby, Clin. Exp. Immunol. 45, 97 (1981); Bausserman et al., op. cit., (1983)). The inherent stickiness of lipophilic proteins causes inaccurate measurements of the proteins in immunoassays.
Three representative lipophilic proteins present in serum and plasma are apolipoprotein Al, apolipoprotein B, and serum amyloid A. Apolipoprotein Al (hereinafter apoAl) is one of the major proteins present in high-density lipoproteins (hereinafter HDL). Apo Al may be a necessary structural component of HDL (H. K. Naito, J. Clin. Immunoassay 9, 11 (1986), and it is an activator of lecithin cholesterol acetyl transferase, an enzyme in the pathway which removes cholesterol from peripheral blood. Apolipoprotein B (hereinafter apoB), the principal protein constituent of low density lipoproteins (hereinafter LDL), is active in recognition of cellular receptors for catabolism of LDL. Serum amyloid A (hereinafter SAA) is also associated with HDL, but this lipoprotein plays no known role in lipid transport. SAA is one of the acute phase reactants, i.e., it is present at elevated levels during acute inflammatory states.
ApoAl consists of a single unglycosylated chain of 243 to 245 amino acid residues, which do not include cystine, cysteine, or leucine. Several isotypes of ApoAl exist, and the lipid-free state of the protein has an alpha helical content of 55% which increases to 75% when phoppholipid is bound to the apoprotein. ApoAl is synthesized in liver and intestine.
The clinical importance of ApoAl measurements lies in its utility in assessing coronary artery disease. As stated above, ApoAl levels are decreased in individuals with coronary disease and are therefore of clinical significance. ApoB levels, in contrast, are elevated in coronary disease, and comparative measurement of ApoAl and ApoB levels provides a sensitive clinical profile.
The concentration of SAA in plasma and other biological specimens is also of clinical significance (Rosenthal and Franklin, J. Clin. Invest. 55, 746 (1975); Gorevic et al., Clin. Immunopathol. 6, 83 (1976); Pepys & Baltz, op. cit., (1983); Sipe, in Laboratory Diagnostic Procedures in the Rheumatic Diseases, A. S. Cohen, ed., Grune and Stratton, Orlando (1985), p. 77; Kushner & Mackiewicz, Disease Markers 5, 1 (1986)). There is minimal SAA synthesis during homeostasis, but within a few hours after injury, two major and several minor isoforms of SAA can be detected in plasma high density lipoproteins (hereinafter HDL) (Benditt and Eriksen, Proc. Natl. Acad. Sci. USA 74, 4925 (1977); Bausserman et al., J. Exp. Med. 152, 641 (1980); Benditt et al., Meth. Enzymol. 163, 510 (1988); Strachan et al., J. Biol. Chem. 264; 18368 (1989)). The amount and duration of SAA production during the acute phase response to tissue injury and cell necrosis depend upon the type of injury and its magnitude (McAdam et al., J. Clin. Invest. 61, 390 (1978); Sipe, in Rheumatology and Immunology, A. S. Cohen and J. C. Bennett, eds., Grune and Stratton, Orlando (1986), p. 97).
Synthesis of SAA is regulated by secretory products of the macrophase such as interleukin-1, tumor necrosis factor, and interleukin-6 (Vogel and Sipe, Surv. Immunol. Res. 1, 235 (1982); Ganapathi et al., Biochem. Biophys. Res. Commun. 157, 271 (1988)). SAA is cleared and/or consumed from plasma more rapidly than most glycosylated acute phase proteins (L. L. Bausserman, in Amyloidosis, J. Marrink and M. H. vanRijswijk, eds., Martinus Nijhoff, Amsterdam (1986), p. 337). SAA concentration is thus a useful indicator of the recent production and action of Il-1 and related cytokines.
In the past two decades, SAA has been studied as the precursor of amyloid fibrils, as an apoprotein constituent of HDL, and, most extensively, as an acute phase protein. Because the concentration of circulating SAA is a sensitive, specific and quantitative marker of recent tissue damage and cell necrosis, it is of interest to monitor SAA values in clinical practice. However, despite its potential usefulness, reliable clinical measurement of SAA and many other lipophilic proteins has not been possible. This is in large part due to the physicochemical properties of these proteins.
There are two amphipathic helical regions in SAA, one in the amino terminal portion of the molecule spanning residues 1-24, and the second from residues 50 to 74 (Parmelee et al, Biochemistry 21, 3298 (1982)). Dramatic changes in conformation and solubility occur when the carboxyl portion of some isoforms of the SAA molecule is removed by proteolytic cleavage to form amyloid A (AA). AA protein forms insoluble fibrils having the cross beta pleated sheet conformation and accumulating in the extracellular spaces of tissues (Benditt and Eriksen, J. Pathol. 65, 231 (1971); Glenner, N. Eng. J. Med. 302, 1283 (1980)). The isolated fibrils are reported to be minimally antigenic and immunogenic (Ram et al., Int. Arch. Allergy 34, 269 (1968)).
Although the major portion of SAA in plasma may be isolated with HDL proteins after hours of centrifugation in the presence of high concentrations of salt, it has been reported that a portion of plasma SAA is present in the nonlipoprotein fraction of plasma (Marhaug, et al., Clin. Exp. Immunol. 50, 382 (1982), Bausserman, personal communication). Because it is not bound to lipids, the SAA in the nonlipoprotein plasma fraction can be expected to have a different conformation from that isolated with HDL proteins. A plasma sample could thus conceivably contain both SAA conformers, i.e., free SAA and HDL-associated SAA. Conformational differences between the conformers can affect the accuracy of immunological assays, since a particular antibody or antiserum may not recognize all of the epitopes exposed in the various conformers. Similar phenomena may also occur because of the existence of more than one isotype of SAA present in the same individual.
The technical difficulties surrounding accurate, reproducible quantification of SAA concentration in plasma and other biological fluids have been widely noted (Marhaug, Scand. J. Immunol. 18, 329 (1983); Pepys and Baltz, op. cit., (1983); Godenir et al., J. Immunol. Methods 83, 217 (1985); Benditt et al., op. cit., (1988)). It has often been reported that optimal immunochemical measurement requires denaturation and dissociation of SAA from lipids and apolipoproteins. Some laboratories have found that denaturation by heat, acid or alkali increase immunoreactivity and reproducibility of SAA measurements (Sipe et al., op. cit., (1976); van Rijswijk, Amyloidosis, Ph.D. Thesis, University of Gronigen, The Netherlands (1981); Eriksen and Benditt, op. cit., (1986). On the other hand, other investigators report that the use of denaturing treatments results in less satisfactory quantification (Benson and Cohen, Arthritis Rheum. 22, 36 (1979); Chambers and Whicher, J. Immunol. Methods 59, 95 (1983); Marhaug, op. cit. (1983)). The basis for these differing observations is thought to lie in the epitope specificity of the antibodies employed and in the particular type of immunoassay employed (direct or competitive binding radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA) or radial immunodiffusion).
Purified apoSAA may be measured by any of the traditional methods for measuring proteins. However, methods such as gel scanning, amino acid analysis, or high performance liquid chromatography are expensive and labor intensive and are thus unsuitable for clinical use. Moreover, these methods require purification since serum and plasma contain substances which may interfere with traditional protein assays. Furthermore, measurement of purified SAA may not accurately reflect the levels of the native lipoprotein.
Immunoassays that measure SAA in the native state are highly desirable, since clinical laboratories routinely use these kinds of procedures, and such assays form the basis of a large portion of general clinical literature on many biological ligands. Several immunoassays for SAA have been described (DeBeer et al., 1982, Bausserman et al., 1988, Bensor and Cohen, 1979). However, the known assays of SAA are subject to interference from other plasma constituents and have not proven clinically useful.
Moreover, the known clinically applicable immunoassays are only semi-quantitative, with sensitivities adequate for the high SAA levels observed in acute states such as pneumonia or trauma but inadequate for monitoring SAA in chronic patients. SAA levels in chronic rheumatic patients can be as low as 5-100 .mu.g/ml, and changes in SAA levels of a chronic rheumatic patient as small as 10 to 20 .mu.g/ml can be clinically relevant. Monitoring of chronic patients necessitates, therefore, a more sensitive assay than has previously been available.
Finally, inflammatory stimulation stimulates circulating SAA levels by several hundred to a thousandfold. In order to be clinically useful, therefore, it is highly desirable that an assay for SAA be accurate over a concentration range of several orders of magnitude.
Marhaug (op. cit., 1983) compared a sandwich ELISA using polyclonal rabbit anti-AA and SAA and monoclonal mouse anti-SAA antibodies with an inhibition ELISA in which plates were coated with apoSAA, and test samples were incubated with monoclonal anti-SAA antibodies prior to addition to the wells. Both methods were affected by self-coating with SAA. Denaturation was not required and delipidation resulted in slightly reduced immunoreactivity. Neither assay provided the sensitivity required for clinically relevant measurements of SAA. Marhaug also described a radioimmunoassay, which he found to be more accurate than either ELISA. However, radioimmunoassay has numerous drawbacks inherent to use of radioactivity, and is thus not the method of choice for clinical measurement of SAA.
Dubois and Malmendier (J. Immunol. Methods 112, 71 (1988)) describe double sandwich ELISA methodology for measurement of human apolipoprotein S (probably identical to SAA) that utilized peroxidase conjugated anti-SAA antibodies to quantify apoS bound to wells coated with affinity purified anti-apoSAA. The sensitivity of this assay was adequate. However, the assay is very labor intensive because of the requirement for affinity purification of antibodies and conjugates. Moreover, the expense of the reagents is higher than is desirable for routine clinical use.
Zuckerman and Surprenant (J. Immunol. Methods 92, 37 (1986)) described a method in which SAA was directly coated from mouse serum at 4.degree. C. in bicarbonate buffer pH 9.6. Like most of the previous assays, this assay expresses SAA concentrations in relative units rather than absolute amounts. As indicated above, expression of SAA concentrations in relative units limits comparison of values from samples obtained and assayed over a period of time and limits comparison of results among laboratories. Without such juxtaposition the clinical utility of any SAA assay is severely limited.
The methodology of Zuckerman and Surprenant is relatively quantitative for mouse SAA when samples were assayed at the same dilution. This method can be modified for measurement of SAA in human samples by using SAA-rich lipoprotein fractions to construct standard curves which would be more stable than those obtained with plasma. Samples within a group may then be assayed at the same dilution and the SAA concentration expressed in relative amounts of SAA-rich HDL.
However, the Zuckerman and Surprenant method has not proven suitable for clinical measurement of SAA in human plasma samples. In order to obtain meaningful comparisons, clinical samples must assayed over a SAA concentration range of several orders of magnitude. This necessitates assay of multiple sample dilutions of varying protein concentrations. Human plasma samples contain interfering proteins which affect binding in different ways at different sample dilutions. It is postulated that human SAA may interact more strongly with other plasma constituents than does mouse SAA. The method of Zuckerman and Surprenant contains no corrective provision for the variable interferences found inhuman plasma samples.
Moreover, SAA from human plasma has been found to bind less efficiently to microtiter plates than does SAA from mouse plasma or serum under the Zuckerman and Surprenant conditions. Another complication arises because the samples measured by Zuckerman and Surprenant were from mice which had experienced experimental inflammatory stimulation. Consequently SAA levels in the mice were high as compared with the relatively low SAA concentrations associated with human disease. It is believed that the sensitivity of the modified Zuckerman and Surprenant assay is therefore not sufficient to measure SAA levels in human clinical samples.
Antigen capture systems using a double sandwich solid phase ELISA with polyclonal rabbit anti-human SAA antiserum as the detection antibody have also proven ineffective for measurement of SAA in serum and plasma samples. The double sandwich solid phase ELISA yielded high results which were also variable. See, e.g., Marhaug (op. cit. 1983.) This method immobilizes antibody on casein-blocked microtiter plates and the analyte is added in solution. Moreover, the same amount of SAA bound to the casein-blocked plates whether or not antibody was immobilized on the plates. When some of the same samples were purified and assayed by the modified Zuckerman/Surprenant method, the SAA levels were observed to be much lower than the values from the double sandwich solid phase ELISA. These results suggest that the double sandwich solid phase ELISA for SAA was not accurate because of interactions between the SAA in the sample with the blocking agent, among SAA molecules in the sample, or between SAA molecules and other serum constituents such as albumin, fibronectin, or SAP.
Covalent binding of SAA to Co-Bind plates (Micro Membranes, 95 Orange Street, Newark, N.J. 01720) has also been ineffective, Purified SAA binds efficiently to the Co-Bind plates, showing greater levels of binding at pH 9.6 than at pH 7.2, which suggests that the increase in pH induces conformational changes exposing free amino groups which bound to the plates. Binding of purified SAA to the plates could be blocked, however, nonspecific binding was dramatically increased in plasma samples. Denaturation by heat or guanidine treatment of samples prior to capture was required to maximally expose determinants.
Direct binding of SAA from plasma to polyvinylchloride plates followed by delipidation with organic solvents is not sufficiently sensitive. The observation of Serban (U.S. Pat. No. 4,782,014) that SAA preferentially binds to plastic surfaces in the presence of a large excess of irrelevant protein is of limited utility in perfecting an immunoassay for SAA. Binding of SAA to the plastic surface must be controlled reproducibly and in such a way that a quantity directly proportional to the concentration of SAA in the test sample is bound. Such controlled and reproducible biding was not present in Serban.
Benditt et al. (op. cit., 1988) describe a competitive inhibition ELISA methodology in which plates are precoated with purified AA protein and the heat denatured samples are incubated with affinity purified apo-SAA antibodies. Subtractive competition assays measuring the ability of plasma samples to compete with antibody for binding to SAA antigen coated on plates was not sufficiently sensitive to detect large quantities of antigen and antibody are required for this assay.
Other proteins found in biological fluids have been similarly difficult to assay. IL-1 beta and CRP are examples of such proteins.
Although it has been relatively easy to measure IL-1 beta in culture supernatants by immunoassay, it has been difficult in plasma. Interference by plasma lipids and/or lipoproteins is indicated by Duff's laboratory (Eastgate, J. A., et al., Lancet, p. 706, Sep. 24, 1988) in which chloroform extraction is performed prior to measurement of IL-1 beta in the plasma of rheumatoid arthritis patients by ELISA. Accordingly, the present invention provides an improved assay for IL-1 beta.
The two clinical methods most frequently used for CRP are nephelometry and radial immunodiffusion. Both methods have a threshold of 5-8 .mu.g/ml. The methodology of the present invention provides for measurement of CRP in the range of 1-10 .mu.g/ml which may be clinically useful for rheumatoid arthritis patients.
In order to be useful for quantitative clinical measurements, it is highly desirable that an assay for any protein have the following characteristics:
1. Potential to measure the ligand in essentially absolute amounts rather than relative units. PA1 2. Potential for accurate measurement of the ligand over a clinically relevant concentration range of the ligand; PA1 3. Potential for automation of the assay. PA1 4. Simple, reliable, inexpensive, and nonhazardous. PA1 a. contacting the sample at high salt concentration and elevated temperature with a support medium having an affinity for the adherent protein under conditions to promote binding of the adherent protein to the solid support; PA1 b. contacting the support medium obtained in step (a) with at least one anti-ligand for the adherent protein under conditions to promote binding of the anti-ligand to the adherent protein; PA1 c. detecting and measuring the amount of anti-ligand bound to the adherent protein; PA1 d. either (i) relating the amount of anti-ligand determined in step (c) with the amount of anti-ligand measured for at least one control sample prepared in accordance with steps (a)-(c), said control sample comprising one or more unrelated proteins known to be free of the adherent protein, or (ii) relating the amount of anti-ligand measured in step (c) with the amount of anti-ligand measured for samples containing known amounts of adherent protein, said samples comprising purified adherent protein in a solution of one or more unrelated proteins and prepared in accordance with steps (a)-(c).
The ability to measurement absolute units permits comparison of results from samples obtained and/or measured over a period of time and comparison of results from different laboratories. It is also desirable that the test be capable of performance by multiple laboratory workers of relatively unsophisticated skill levels.
Thus, improved methods for measuring levels of proteins, especially those technically difficult to measure, are being sought because of the deficiencies of present methodologies.